The GAPs, GEFs, and GDIs of heterotrimeric G-protein alpha subunits

The heterotrimeric G-protein alpha subunit has long been considered a bimodal, GTP-hydrolyzing switch controlling the duration of signal transduction by seven-transmembrane domain (7TM) cell-surface receptors. In 1996, we and others identified a superfamily of “regulator of G-protein signaling” (RGS) proteins that accelerate the rate of GTP hydrolysis by Gα subunits (dubbed GTPase-accelerating protein or “GAP” activity). This discovery resolved the paradox between the rapid physiological timing seen for 7TM receptor signal transduction in vivo and the slow rates of GTP hydrolysis exhibited by purified Gα subunits in vitro. Here, we review more recent discoveries that have highlighted newly-appreciated roles for RGS proteins beyond mere negative regulators of 7TM signaling. These new roles include the RGS-box-containing, RhoA-specific guanine nucleotide exchange factors (RGS-RhoGEFs) that serve as Gα effectors to couple 7TM and semaphorin receptor signaling to RhoA activation, the potential for RGS12 to serve as a nexus for signaling from tyrosine kinases and G-proteins of both the Gα and Ras-superfamilies, the potential for R7-subfamily RGS proteins to couple Gα subunits to 7TM receptors in the absence of conventional Gβγ dimers, and the potential for the conjoint 7TM/RGS-box Arabidopsis protein AtRGS1 to serve as a ligand-operated GAP for the plant Gα AtGPA1. Moreover, we review the discovery of novel biochemical activities that also impinge on the guanine nucleotide binding and hydrolysis cycle of Gα subunits: namely, the guanine nucleotide dissociation inhibitor (GDI) activity of the GoLoco motif-containing proteins and the 7TM receptor-independent guanine nucleotide exchange factor (GEF) activity of Ric‑8/synembryn. Discovery of these novel GAP, GDI, and GEF activities have helped to illuminate a new role for Gα subunit GDP/GTP cycling required for microtubule force generation and mitotic spindle function in chromosomal segregation

G protein signaling in the parasite Entamoeba histolytica

The parasite Entamoeba histolytica causes amebic colitis and systemic amebiasis. Among the known amebic factors contributing to pathogenesis are signaling pathways involving heterotrimeric and Ras superfamily G proteins. Here, we review the current knowledge of the roles of heterotrimeric G protein subunits, Ras, Rho and Rab GTPase families in E. histolytica pathogenesis, as well as of their downstream signaling effectors and nucleotide cycle regulators. Heterotrimeric G protein signaling likely modulates amebic motility and attachment to and killing of host cells, in part through activation of an RGS-RhoGEF (regulator of G protein signaling–Rho guanine nucleotide exchange factor) effector. Rho family GTPases, as well as RhoGEFs and Rho effectors (formins and p21-activated kinases) regulate the dynamic actin cytoskeleton of E. histolytica and associated pathogenesis-related cellular processes, such as migration, invasion, phagocytosis and evasion of the host immune response by surface receptor capping. A remarkably large family of 91 Rab GTPases has multiple roles in a complex amebic vesicular trafficking system required for phagocytosis and pinocytosis and secretion of known virulence factors, such as amebapores and cysteine proteases. Although much remains to be discovered, recent studies of G protein signaling in E. histolytica have enhanced our understanding of parasitic pathogenesis and have also highlighted possible targets for pharmacological manipulation

Structural Determinants of Ubiquitin Conjugation in Entamoeba histolytica

Ubiquitination is important for numerous cellular processes in most eukaryotic organisms, including cellular proliferation, development, and protein turnover by the proteasome. The intestinal parasite Entamoeba histolytica harbors an extensive ubiquitin-proteasome system. Proteasome inhibitors are known to impair parasite proliferation and encystation, suggesting the ubiquitin-proteasome pathway as a viable therapeutic target. However, no functional studies of the E. histolytica ubiquitination enzymes have yet emerged. Here, we have cloned and characterized multiple E. histolytica ubiquitination components, spanning ubiquitin and its activating (E1), conjugating (E2), and ligating (E3) enzymes. Crystal structures of EhUbiquitin reveal a clustering of unique residues on the α1 helix surface, including an eighth surface lysine not found in other organisms, which may allow for a unique polyubiquitin linkage in E. histolytica. EhUbiquitin is activated by and forms a thioester bond with EhUba1 (E1) in vitro, in an ATP- and magnesium-dependent fashion. EhUba1 exhibits a greater maximal initial velocity of pyrophosphate:ATP exchange than its human homolog, suggesting different kinetics of ubiquitin activation in E. histolytica. EhUba1 engages the E2 enzyme EhUbc5 through its ubiquitin-fold domain to transfer the EhUbiquitin thioester. However, EhUbc5 has a >10-fold preference for EhUba1∼Ub compared with unconjugated EhUba1. A crystal structure of EhUbc5 allowed prediction of a noncovalent “backside” interaction with EhUbiquitin and E3 enzymes. EhUbc5 selectively engages EhRING1 (E3) to the exclusion of two HECT family E3 ligases, and mutagenesis indicates a conserved mode of E2/RING-E3 interaction in E. histolytica

Structural basis for nucleotide exchange on G i subunits and receptor coupling specificity

Heterotrimeric G proteins are molecular switches that relay information intracellularly in response to various extracellular signals. How ligand-activated G protein-coupled receptors act at a distance to exert exchange activity on the Galpha nucleotide binding pocket is poorly understood. Here we describe the synergistic action of two peptides: one from the third intracellular loop of the D2 dopamine receptor (D2N), and a second, Galpha.GDP-binding peptide (KB-752) that mimics the proposed role of Gbetagamma in receptor-promoted nucleotide exchange. The structure of both peptides in complex with Galpha(i1) suggests that conformational changes in the beta3/alpha2 loop and beta6 strand act in concert for efficient nucleotide exchange. Two key residues in the alpha4 helix were found to define a receptor/Galpha(i) coupling specificity determinant

Entamoeba histolytica Rho1 Regulates Actin Polymerization through a Divergent, Diaphanous-Related Formin

Entamoeba histolytica requires a dynamic actin cytoskeleton for intestinal and systemic pathogenicity. Diaphanous-related formins represent an important family of actin regulators that are activated by Rho GTPases. The E. histolytica genome encodes a large family of Rho GTPases and three diaphanous-related formins, of which EhFormin1 is known to regulate mitosis and cytokinesis in trophozoites. We demonstrate that EhFormin1 modulates actin polymerization through its formin homology 2 (FH2) domain. Despite a highly divergent diaphanous autoinhibitory domain, EhFormin1 is autoinhibited by an N- and C-terminal intramolecular interaction, but activated upon binding of EhRho1 to the N-terminal domain tandem. A crystal structure of the EhRho1·GTPγS/EhFormin1 complex illustrates an EhFormin1 conformation that diverges from mammalian mDia1 and lacks a secondary interaction with a Rho insert helix. The structural model also highlights residues required for specific recognition of the EhRho1 GTPase and suggests that the molecular mechanisms of EhFormin1 autoinhibition and activation differ from mammalian homologs

A Role for Regulator of G Protein Signaling-12 (RGS12) in the Balance Between Myoblast Proliferation and Differentiation

Regulators of G Protein Signaling (RGS proteins) inhibit G protein-coupled receptor (GPCR) signaling by accelerating the GTP hydrolysis rate of activated Gα subunits. Some RGS proteins exert additional signal modulatory functions, and RGS12 is one such protein, with five additional, functional domains: a PDZ domain, a phosphotyrosine-binding domain, two Ras-binding domains, and a Gα·GDP-binding GoLoco motif. RGS12 expression is temporospatially regulated in developing mouse embryos, with notable expression in somites and developing skeletal muscle. We therefore examined whether RGS12 is involved in the skeletal muscle myogenic program. In the adult mouse, RGS12 is expressed in the tibialis anterior (TA) muscle, and its expression is increased early after cardiotoxin-induced injury, suggesting a role in muscle regeneration. Consistent with a potential role in coordinating myogenic signals, RGS12 is also expressed in primary myoblasts; as these cells undergo differentiation and fusion into myotubes, RGS12 protein abundance is reduced. Myoblasts isolated from mice lacking Rgs12 expression have an impaired ability to differentiate into myotubes ex vivo, suggesting that RGS12 may play a role as a modulator/switch for differentiation. We also assessed the muscle regenerative capacity of mice conditionally deficient in skeletal muscle Rgs12 expression (via Pax7-driven Cre recombinase expression), following cardiotoxin-induced damage to the TA muscle. Eight days post-damage, mice lacking RGS12 in skeletal muscle had attenuated repair of muscle fibers. However, when mice lacking skeletal muscle expression of Rgs12 were cross-bred with mdx mice (a model of human Duchenne muscular dystrophy), no increase in muscle degeneration was observed over time. These data support the hypothesis that RGS12 plays a role in coordinating signals during the myogenic program in select circumstances, but loss of the protein may be compensated for within model syndromes of prolonged bouts of muscle damage and repair

G-protein Signaling Modulator-3 Regulates Heterotrimeric G-protein Dynamics through Dual Association with Gβ and Gα i Protein Subunits

Regulation of the assembly and function of G-protein heterotrimers (Gα·GDP/Gβγ) is a complex process involving the participation of many accessory proteins. One of these regulators, GPSM3, is a member of a family of proteins containing one or more copies of a small regulatory motif known as the GoLoco (or GPR) motif. Although GPSM3 is known to bind Gαi·GDP subunits via its GoLoco motifs, here we report that GPSM3 also interacts with the Gβ subunits Gβ1 to Gβ4, independent of Gγ or Gα·GDP subunit interactions. Bimolecular fluorescence complementation studies suggest that the Gβ-GPSM3 complex is formed at, and transits through, the Golgi apparatus and also exists as a soluble complex in the cytoplasm. GPSM3 and Gβ co-localize endogenously in THP-1 cells at the plasma membrane and in a juxtanuclear compartment. We provide evidence that GPSM3 increases Gβ stability until formation of the Gβγ dimer, including association of the Gβ-GPSM3 complex with phosducin-like protein PhLP and T-complex protein 1 subunit eta (CCT7), two known chaperones of neosynthesized Gβ subunits. The Gβ interaction site within GPSM3 was mapped to a leucine-rich region proximal to the N-terminal side of its first GoLoco motif. Both Gβ and Gαi·GDP binding events are required for GPSM3 activity in inhibiting phospholipase-Cβ activation. GPSM3 is also shown in THP-1 cells to be important for Akt activation, a known Gβγ-dependent pathway. Discovery of a Gβ/GPSM3 interaction, independent of Gα·GDP and Gγ involvement, adds to the combinatorial complexity of the role of GPSM3 in heterotrimeric G-protein regulation

Cortical localization of the G protein GPA-16 requires RIC-8function during C. elegans asymmetric cell division

Understanding of the mechanisms governing spindle positioning during asymmetric division remains incomplete. During unequal division of one-cell stag

A P-loop Mutation in Gα Subunits Prevents Transition to the Active State: Implications for G-protein Signaling in Fungal Pathogenesis

Heterotrimeric G-proteins are molecular switches integral to a panoply of different physiological responses that many organisms make to environmental cues. The switch from inactive to active Gαβγ heterotrimer relies on nucleotide cycling by the Gα subunit: exchange of GTP for GDP activates Gα, whereas its intrinsic enzymatic activity catalyzes GTP hydrolysis to GDP and inorganic phosphate, thereby reverting Gα to its inactive state. In several genetic studies of filamentous fungi, such as the rice blast fungus Magnaporthe oryzae, a G42R mutation in the phosphate-binding loop of Gα subunits is assumed to be GTPase-deficient and thus constitutively active. Here, we demonstrate that Gα(G42R) mutants are not GTPase deficient, but rather incapable of achieving the activated conformation. Two crystal structure models suggest that Arg-42 prevents a typical switch region conformational change upon Gαi1(G42R) binding to GDP·AlF4− or GTP, but rotameric flexibility at this locus allows for unperturbed GTP hydrolysis. Gα(G42R) mutants do not engage the active state-selective peptide KB-1753 nor RGS domains with high affinity, but instead favor interaction with Gβγ and GoLoco motifs in any nucleotide state. The corresponding Gαq(G48R) mutant is not constitutively active in cells and responds poorly to aluminum tetrafluoride activation. Comparative analyses of M. oryzae strains harboring either G42R or GTPase-deficient Q/L mutations in the Gα subunits MagA or MagB illustrate functional differences in environmental cue processing and intracellular signaling outcomes between these two Gα mutants, thus demonstrating the in vivo functional divergence of G42R and activating G-protein mutants

Receptor-selective Effects of Endogenous RGS3 and RGS5 to Regulate Mitogen-activated Protein Kinase Activation in Rat Vascular Smooth Muscle Cells

Regulators of G protein signaling (RGS) proteins compose a highly diverse protein family best known for inhibition of G protein signaling by enhancing GTP hydrolysis by Galpha subunits. Little is known about the function of endogenous RGS proteins. In this study, we used synthetic ribozymes targeted to RGS2, RGS3, RGS5, and RGS7 to assess their function. After demonstrating the specificity of in vitro cleavage by the RGS ribozymes, rat aorta smooth muscle cells were used for transient transfection with the RGS-specific ribozymes. RGS3 and RGS5 ribozymes differentially enhanced carbachol- and angiotensin II-induced MAP kinase activity, respectively, whereas RGS2 and RGS7 ribozymes had no effect. This enhancement was pertussis toxin-insensitive. Thus RGS3 is a negative modulator of muscarinic m3 receptor signaling, and RGS5 is a negative modulator of angiotensin AT1a receptor signaling through G(q/11). Also, RGS5 ribozyme enhanced angiotensin-stimulated inositol phosphate release. These results indicate the feasibility of using the ribozyme technology to determine the functional role of endogenous RGS proteins in signaling pathways and to define novel receptor-selective roles of endogenous RGS3 and RGS5 in modulating MAP kinase responses to either carbachol or angiotensin